Embryonic stem cells are a group of undifferentiated totipotential cells derived from inner cell mass of early development stage embryo in fertilized ovum or from primordial germ cells after embryo implantation, have unlimited multiplication and differentiation potential, and can be differentiated into cells of almost all histologic types. Hence, they have a broad prospect in aspects of animal cloning, fundamental research of development biology, especially human regenerative medicine. However, there are still many difficulties in really using embryonic stem cells in clinic; specifically, the resource of human embryonic stem cells especially patient-specific stem cells and related ethical issues are serious challenges in scientific community. Hence, it is an objective pursued and struggled by many researchers to obtain pluripotent cells similar to embryonic stem cells from undifferentiated cells by using abundant somatic cells via reprogramming methods.
So far, there are mainly three techniques for obtain stem cells by inducing reprogramming in somatic cells: somatic cell nuclear transfer reprogramming (SCNT), cell fusion reprogramming and induced pluripotent stem cell reprogramming (iPS). In 1952, nuclear transfer was firstly obtained successfully in amphibians (Briggs and King, 1952), the clone sheep Dolly was born in 1997 (Wilmut et al., 1997), and somatic cell cloning technique rapidly developed and gradually matured. So far, somatic cell cloning has been successfully implemented in more than 20 animals such as rats, pigs, bovines, monkeys and dogs. Stem cells obtained by nuclear transfer can avoid adverse reactions such as immunological rejection after cell transplantation therapy.
However, this technique still has many problems in real clinical applications. Firstly, nuclear transfer has a very low efficiency; secondly, some experiments confirm that somatic cell cloning animals frequently have abnormal development problem; and sources of human ovum and use of human embryo associated to final applications in treatment of human diseases are still in ethical controversy. All these are bottleneck problems for this technique. Similarly, cell fusion reprogramming technique also faces many problems such as very low reprogramming efficiency, too high requirement in technology, which restrict the clinical applications thereof. Both of the above two conventional reprogramming methods have drawbacks, so that many scientists in the world are exploring other more feasible reprogramming strategies. In 2006, the research group of Japan scientist Yamanaka found that 4 transcription factors Oct4, Sox2, Klf4 and c-Myc could be transferred into mouse fibroblasts via viral infection, then the obtained fibroblasts had pluripotency similar to that of ES cells (Takahashi and Yamanaka, 2006). The subsequent researches showed that such induced pluripotent stem cells (iPS cells) were very similar to embryonic stem cells and could form chimeric mice after being injected into blastula. In particular, the birth of mice generated via tetraploid complementation technique in 2009 confirms the pluripotency of this kind of cells. In November of 2007, the laboratories of Yamanaka and Thomson separately declared that they successfully induced human iPS cells by using human skin cells (Takahashi et al., 2007; Yu et al., 2007). In the same year, the research group of Jaenisch achieved primary success in gene therapy by using iPS technique in sickle cell anemia mouse model. In brief, reprogramming and recovery of pluripotency surprisingly occurred in differentiated somatic cells by introducing several simple transcription factors via this technique. This simple but feasible technique breakthrough can conveniently obtain pluripotent stem cells from somatic cells of patients themselves, which not only simply solves the problem of cell sources for regeneration therapy, but also avoids autoimmune rejection, evades ethic restriction, and establishes solid basis for clinical application of regeneration medicine.
However, the technique for inducing pluripotent stem cells as a new technique is imperfect in many aspects. For example, its mechanism is not clear, it may have potential risks in safety, and it has low induction efficiency and a long induction time. If these problems could not be sufficiently solved, this technique cannot be successfully used in clinic. Hence, tremendous efforts have been made to solve these problems, and a lot of progresses have been achieved so far.
Firstly, scientists in the world have made sufficient researches in iPS induction mechanism from aspect of molecular biology and molecular biology. For example, the scientists' articles in terms of single cell level, chromatin modification enzyme and 3D chromatin regulation give us in-depth knowledge of reprogramming mechanism in transcription level, epigenetic level, signal transduction and so on. In particular, the “seesaw model” of Hongkui Deng of Peking University lets us know the iPS mechanism more comprehensively.
Secondly, many improvements have been made in safety of iPS. First of all, c-Myc was removed from the 4 factors so as to significantly reduce risks in tumorigenicity. In addition, more safe induction means, such as use of non-viral integration vectors, mRNA, protein induction means, small molecule induction means of Hongkui Deng, make great improvement in safety of iPS.
Thirdly, conventional iPS has an induction efficiency of about 0.01% to 2%. Thus, many scientists use various methods to improve induction efficiency. For example, the addition of small molecular compounds such as VPA, VC can elevate induction efficiency by about 100 times, and the optimized combination of induction factors such as mRNA induction may elevate the efficiency up to about 5%. Pluripotent factor fused VP16 or transcriptional activation domain of MyoD may also significantly increase iPS induction efficiency.
Fourthly, the iPS induction time for mouse cells is about 2 weeks in general, and the time of human iPS cells is much longer. Thus, it is also a very important factor for final clinical application to obtain iPS in the shortest possible time, but at present, the induction time is usually about 2 weeks.
In the patent application with application number of WO2011110051, OCT4, SOX2, NANOG are separately fused with herpes virus VP16 transcription activation domain. These 3 kinds of artificial transcription factors together with Klf4 infect MEF cells, which may significantly improve reprogramming efficiency. However, this technique still does not achieve ideal conditions in terms of speed and efficiency. For example, the expression of endogenous pluripotent genes such as Oct4 is not fast enough. Hirai et al. fused OCT4 and MyoD transcription activation domain, then the MyoD-fused OCT4 as an artificial factor (M30) together with three transcription factors, SOX2, c-Myc, Klf4 in primitive form are used to infect MEF cells, the reprogramming efficiency was significantly elevated. GFP positive clone count result showed this method for inducing pluripotent stem cells could achieve the highest value of GFP positive clone on the day 15, and the induction efficiency was up to about 25%, which was about 10 times that of conventional Oct4, SOX2, c-Myc, Klf4 induction method. In the meantime, this method still uses proto-oncogene c-Myc, and thus cannot avoid potential safety risk. In comparison, the present invention has higher safety, higher efficiency, and shorter induction time. Thus, the present invention is more promising in regenerative medicine clinic application in future.
Although the researches of iPS have achieved considerable progresses in the past several years and its glorious prospect in final clinical uses is gradually revealed to us, the technique in general still has problems such as in low induction efficiency, long time, and safety problem, which impede rapid and efficient acquisition of high quality iPS cells for clinical application.